Method of treating inflammation with antibodies to neutrophil chemotactic factor

ABSTRACT

An isloated, synthetic preparation of a novel neutrophil-specific chemotactic factor (NCF), monoclonal antibodies having specific binding affinity for NCF and a clone containing the complete cDNA coding sequence for NCF are disclosed.

This is a divisonal of application Ser. No. 08/467,612 filed Jun. 6,1995, now U.S. Pat. No. 5,698,196, which is a divisional of applicationSer. No. 07/169,033, filed Mar. 16, 1988, now U.S. Pat. No. 5,652,338.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to an isolated, synthetic preparation of anovel neutrophil-specific chemotactic factor (NCF), monoclonalantibodies having specific binding affinity for NCF and a clonecontaining the complete coding sequence for NCF.

2. State of the Art

Activated monocytes/macrophages produce various mediators that causeinflammation. Among them are chemotactic factors which cause white bloodcells to migrate into inflammatory sites where these factors areareleased. Neutrophils, the dominant leukocytes attracted by thechemotactic factors, are believed to play a critical role in theinflammatory reactions. Such diseases as rheumatoid arthritis,idiopathic pulmonary fibrosis and certain pathological inflammatorychanges in many other conditions are believed to be caused byneutrophils and/or their products. However, a specific pro-inflammatorymediator released by tissue macrophages and other cells in response toimflammatory stmuli and leading to neutrophil-rich leukocyteaccumulation in host defense and disease, has not heretofore beenidentified and isolated.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide abiologically active novel synthetic polypeptide acting as aneutrophil-specific chemotactic factor (NCF).

It is a further object of the present invention to provide a molecularclone containing the complete coding sequence for the synthesis of NCFby either prokaryotic or eukaryotic expression vectors.

It is a still further object of ,the present invention to providemonoclonal antibodies having specific binding affinity for NCF of thepresent invention.

It is another object of the present invention to provide a kitcomprising a container containing the cDNA for NCF quantitation,detection or localization of NCF mRNA in a body sample.

It is yet another object of the present invention to provide a kitcomprising a container containing anti-NCF antibodies having specificbinding affinity for NCF for quantitation, detection or localization ofNCF in a body sample.

Other objects and advantages will become evident from the followingdetailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantagesof the invention will be better understood upon a reading of thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

FIG. 1 demonstrates translation of cDNA into NCF protein in reticulocytelysate system; and

FIGS. 2A-2D shows:

(a) Northern blot analysis of mRNA induction in lipopolysaccharide (LPS)stimulated peripheral blood monomuclear cells (PBMC);

(b) The time course of the accumulation of neutrophil chemotacticactivity in culture media of PBMC after stimulation with LPS;

(c) Induction of NCF mRNA in PBMC by IL 1 or TNF, but not by IL 2,gamma-IFN, and alpha-IFN;

(d) HPLC gel filtration analysis of IL 1 and TNF induced neutrophilchemotactic activity.

DETAILED DESCRIPTION OF THE INVENTION

The above and various other objects and advantages of the presentinvention are achieved by a homogeneously pure, isolated, syntheticneutrophil chemotactic protein, designated herein NCF, composed in thewhole or in part only of the following amino acid sequence (singleletter code):

NH₂ -S-A-K-E-L-R-C-Q-C-I-K-T-Y-S-K-P-F-H-P-K-F-I-K-E-L-R-V-I-E-S-G-P-H-C-A-N-T-E-I-I-V-K-L-S-D-G-R-E-L-C-L-D-P-K-E-N-W-V-Q-R-V-V-E-K-F-L-K-R-A-E-N-S

Unless defined otherwise, all technica1 and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference. Unless mentioned otherwise, thetechniques employed herein are standard methodologies well known to oneof ordinary skill in the art.

Chemical synthesis of the NCF of the present invention composed of the72 amino acid residues as shown above, is achieved by commerciallyavailable polypeptide synthesizers. Alternatively, the NCF of thepresent invention is synthesized by standard techniques employing anexpression vector containing in its genome the cloned complete codingsequence of NCF. Anti-NCF monoclonal antibodies of the present inventionare prepared by standard hybridoma technology and utilized forpurification and assaying purposes following standard immunologicalmethodologies well known in the art.

High performance liquid chromatography (HPLC), in situ hybridizationassays, Northern blotting analyses and the like are typical examples ofthe standard conventional techniques well known to one of ordinary skillin the art, which can be employed for isolation, localization,differentiation, detection, or measurement of the mRNA for NCF inbiological samples.

It should be noted that the fact that chemically synthesized polypeptideof the present invention at 10 nanomolar concentrations acts as aneutrophil attracting factor, is shown by the results presented in Table1.

                  TABLE 1    ______________________________________    Chemotactic response of human    neutrophils to chemically synthesized NCF.    Concentration of                   Percentage of assay    NCF, nanomolar neutrophils that migrated    ______________________________________    1000           23    100            34    10             32    1              5    0.1            1    Hanks medium   0.3    10-.sup.7 M fMet-Leu-Phe                   40    ______________________________________     1. It is typical for chemoattractant doseresponse curves to show an     optimum, with a decreased response at concentrations above the optimum.     2. fMetLeu-Phe is a commonly used reference chemoattractant.

It may be pointed out that various stimuli cause the release orsecretion of more than one chemoattractant. Without the cDNA of thepresent invention, it is clear, of course, that the presence,specificially of the mRNA for NCF as an involved factor in a particularclinico-pathological situation, could not be definitively identified anddiagnosed. cDNA of the present invention due to its binding affinity formRNA for NCF, for the first time makes it possible to analyze bodysamples such as joint fluid, sputum, alveolar lavage fluid, tissuesamples and the like to detect the presence or absence of mRNA for NCF.Of course, the antibodies can also be utilized for diagnostic purposesto detect the NCF and to neutralize the NCF for alleviating any diseaseor anomalous conditions in which the presence of NCF is found to be acausative factor.

A pharmaceutical composition for use in treating inflammatory conditioncomprises and anti-inflammatory effective amount of the anti-NCFmonoclonal antibodies in pharmaceutically acceptable carrier, such asphysiological saline, sterile non-toxic buffer and the like.

A deposit of cDNA for NCF and of the hybridoma for anti-NCF monoclonalantibodies have been made at the ATCC, 10801 University Boulevard,Manassas, Va. 20110-2209 on Jan. 12, 1988 and Feb. 17, 1988,respectively, under the accession numbers 40412 and HB9647,respectively. The deposits shall be viably maintained, replacing if theybecame non-viable, for a period of 30 years from the date of thedeposit, or for 5 years from the last date of request for a sample ofthe deposit, which is longer, and made available to the public withoutrestriction in accordance with the provisions of the law. TheCommissioner of Patents and Trademarks, upon request, shall have accessto the deposits.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

What is claimed is:
 1. A method of treating an inflamrnatory condition,said method comprising administering to a host inflicted with aninflammnatory condition caused by or related to neutrophil chemotacticfactor (NCF) polypeptide, an antiinflammatory effective amount ofanti-NCF antibodies, wherein said antibodies have specific bindingaffinity for a neutrophil chemotactic factor polypeptide or part thereofwhere the polypeptide comprises the following amino acid sequencerepresented by single letter code;NH₂-S-A-K-E-L-R-C-Q-C-I-K-T-Y-S-K-P-F-H-P-K-F-I-K-E-L-R-V-I-E-S-G-P-H-C-A-N-T-E-I-I-V-K-L-S-D-G-R-E-L-C-L-D-P-K-E-N-W-V-Q-R-V-V-E-K-F-L-K-R-A-E-N-S.2. The method of claim 1, wherein said antibodies are monoclonalantibodies.